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Posted: June 1st, 2022
Study on the effect of ethanolic extract of clove on some chemical, organoleptic and microbial properties of chicken fillet at refrigeration temperature
Abstract
Cloves are the aromatic flower buds of a tree Syzygium aromaticum and being rich in polyphenolic compounds. The aim of this study was using of clove extract in different concentrations on the shelf life of chicken fillet at refrigerated temperature. Assessment of chemical spoilage indexes (such as pH, Peroxide Value, Thiobarbituric acid and TVN), microbial quality (Total plate count and Psychrotrophic count) and sensory properties of chicken fillet (smell, texture, taste, color and total acceptability) were done. The results showed a significant increase in chemical and microbiological properties of treated samples with clove extract in comparison with controls (P<0/01). The highest sensory evaluated scores assessed in this research for 3% clove extract treatment. As a consequence, clove extract might be considered as an effective tool in preventing the quality degradation of chicken fillet, resulting in an extension of their shelf life.
Introduction:
Poultry meat is providing an almost perfect medium for microbial growth (Khanjari et al., 2013). Meat is used for its unique taste and is a good source of nutrients, providing animal proteins, fatty acids, minerals, some elements and vitamins (Singh et al., 2014). The color of meat is an important factor more than any other quality factor because it is an indicator of wholesomeness and freshness (Mancini and Hunt, 2005). Some of the important factors for consumer acceptance and the shelf life of good meat are flavor, color, lipid oxidation and microbial growth. One of the major for the deterioration of meat is lipid oxidation that affects its nutritional value, color, flavor, and odor. The decrease in nutritional value during lipid oxidation can be due to loss of essential fatty acids and some vitamins, and production of toxic compounds such as malonaldehyde. There are a lot of factors can influence lipid oxidation like light, temperature, concentration of oxygen, presence of antioxidants, enzymes, pro-oxidants, metal ions such as iron, haem pigments, mechanical processes and etc. (Biswas et al., 2012). Antioxidant and antimicrobial agents are used for preventing of mentioned problems. There are various synthetic chemicals such as tertiary butyl hydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and etc. (Valencia et al., 2007). In order to harmful effects on human health, the use of these synthetic compounds is quite doubtful.
Todays, scientists focus on the search for natural replacement having both antioxidant and antimicrobial activities such as clove, rosemary, ginger, garlic, green tea, cloudberry, beetroot, willow herb, cinnamon and etc., which can maintain meat quality, prevent economic loss, extend shelf life and deliver a good product to consumers. Clove as a rich source of natural compound is dried bud of Syzygium aromaticum and being rich in polyphenolic compounds and have antioxidant and antimicrobial activity due to its main ingredient eugenol (Singh et al., 2014).
There are no published data regarding the use of ethanolic clove extract for enhancing the shelf life of poultry meat especially chicken fillet. Therefore, the objective of this study was to investigate the effect of ethanolic extract of clove on some chemical, organoleptic and microbial properties of the chicken fillet at refrigeration temperature.
2. Materials and methods:
2.1. Clove ethanolic extraction
50 grams of dried clove powder were macerated with 500 ml of ethanol 70% (v/v) under constant mechanical agitation on a rotary shaker at room temperature and protected from light for 48 h. the extract was then filtered and the solvent removed by rotary evaporation at 60˚C. The remaining extract was finally dried in a vacuum oven at 30˚C for two hours to ensure the removal of any residual solvent. The extract was kept in a dark bottle in the refrigerator at 4˚C until use (Kim et al., 2013; Tavassoli and Djomeh, 2011).
2.2. Analysis of volatile compounds of extract by GC/MS
The volatile compounds of extract were isolated by the hexane solvent to inject to GC/MS. GC analyses were performed using a Hewlett Packard GC- HP-6890 gas chromatograph equipped with a FID and a methyl silicon-cross link coloum ( HP-1MS) (60 m·0.20 mm i.d., film thickness 0.25 µm). Oven temperature was programmed by initial temperature 160 ˚C, final temperature 230 ˚C with temperature gradient 7c/min. The injector temperature was 250 ˚C. The carrier gas, helium, was adjusted to a linear velocity of 1ml/min. The sample was injected into the GC by split mode with a split ratio of 1/20. GC/MS analyses were performed using Hewlett Packard-HP-5970. Ion source temperature was 250 ˚C; the ionization energy was 70 eV with a scan time of 1s. Compounds were identified by matching their mass spectra and retention times with those of pure compounds whenever possible. NIST (National Institute of Standards and Technologies) Mass Spectra Library was also used as a reference (Xie et al., 2013).
2.3. Samples preparation
Chicken fillet samples (50 gr) were immersed in ethanolic clove extract at concentrations of 0, 1, 2 and 3 percent. Then the samples packed tightly in plastic bags and kept at refrigerator temperature (4˚C ± 1˚C) for 10 days.
2.4. Chemical analysis of fillets
2.4.1. Protein, fat and moisture content
Protein, fat and moisture content of the chicken fillet were determined according to AOAC (2005). The pH value was recorded by a digital pH meter (Multiline P4WTW).
2.4.2. Peroxide value
Peroxide value of chicken fillet samples was determined using the method described by Shantha and Decker (1994). In fact, peroxide value was measured by IDF standard method, 74A:1991.
2.4.3. TVN
Volatile nitrogen compounds of samples were analyzed by the macro-Kjeldahl method. Total volatile nitrogen of chicken fillet samples was determined using the method described by Parvaneh (1998).
2.4.4. Evaluation of thiobarbituric acid
Lipid oxidation in the chicken fillet was monitored by measuring thiobarbituric acid reactive substances at 0, 3 and 7 days during refrigerated storage. The production of TBA was measured Spectrophotometrically at 532 nm. Thiobarbituric acid number (TBA, mg malondialdehyde/kg) of the chicken fillet samples was determined using the extraction method described by Natseba et al. (2005).
2.5. Microbial analysis
2.5.1. Aerobic Plate Count (APC)
Microbiological counts were determined by placing a 10 gr of samples in 90 ml of buffered peptone solution and homogenized. From this dilution, other decimal dilutions were prepared and plated in the appropriate media. Meanwhile, the Aerobic Plate Count (APC) was determined using plate count agar (PCA, Merck) after incubation for 48h at 35˚C the microbiological data were transformed into logarithms of the number of colonies forming units per gram (Evancho et al., 2001).
2.5.1. Psychrotrophic Bacteria Count
Psychrotrophic counts (PTC) were determined in a similar method to that for APC except that plates were incubated at 7˚C for 10 days (Evancho et al., 2001).
2.6. Sensory evaluation
Sensory evaluation was performed only on uninoculated samples. The attributes of cooked samples (i.e. odor, taste, color, tissue and total accept) were evaluated by using a 6 member trained panel. Treated chicken fillet microwaved and immediately presented to the panelists. The panelist scored attributes by using a 5-point hedonic scale, where 1=unacceptable, 5= very acceptable, while the limit of acceptability was 3. Sensory evaluation was accomplished at 1 day of sampling (Mahdavian Mehr et al., 2010; Zakipour Rahimabadi et al., 2013).
2.7. Statistical analysis
All analyses were carried out in triplicate and mean values ± standard deviations were reported for each case. The one-sample Kolmogorov-Smirnov test was employed to determine the normal distribution of data. Analysis of data among all treatments of clove extract was done by one-way ANOVA (SPSS 22). To determine significant differences between treatments Duncan’s Multiple Range test were used. Friedman test was performed to evaluate the significance of differences among sensory values. A probability level of P<0.01 was used in testing the statistical significance of all experimental data.
3. Results and Discussion
3.1. Analysis of volatile compounds of extract by GC/MS
The GC results showed that the highest percentage compounds were eugenol (84.5%), eugenol acetate (2.2%) and heptacozane (1.6%) respectively. However, the results of chemical compounds in extracts of cloves grown in different regions, extracts from different solvents, is different (Table 1) (Kasai et al., 2015; Nassar et al., 2007).
| Table 1. Constituents of the extract, the amount of them and their retention time | ||
| Percentage | Retention time(min) | The name of compound |
| 1.0 | 11.10 | Decane |
| 0.4 | 12.46 | Phenol,2-methoxy |
| 0.4 | 15.75 | Naphthalene,decahydro-1,5-dimethyl |
| 1.2 | 16.03 | Chavicol |
| 84.5 | 17.59 | Eugenol |
| 0.4 | 19.07 | Trans-Beta-Caryophyllene |
| 2.2 | 19.98 | Eugenyl Acetate |
| 0.3 | 22.49 | 2′ , 3′ ,4′ Trimethoxyacetophenone |
| 1.6 | 23.51 | Heptacosane |
3.2. Chemical analysis of fillets
The moisture, fat and protein content of chicken fillets were 2%, 18.75 %, and 73.2%, respectively.
The pH value:
The results from pH value changes from treatments during refrigeration are shown in Table 2. The pH value during storage chicken fillet was under the influence of changes in nitrogenous compounds and different bacterial activity (Michalczyk et al., 2012). Decomposition of nitrogen compounds during the maintenance period would lead to increase in pH of chicken fillet, which is part of this increase may be related to the growth of proteolytic bacteria (Hulankova et al., 2013; Tajkarimi et al., 2010). The pH values gradually increased with time following increasing concentration of extract. In the present study trend of pH changes was similar to the work of Hulankova et al. (2013). The initial pH was 5.97±0.04 and increased to 6.87± 0.03 by the end of storage at 4˚C. The pH change was increased because of bacterial proteolytic activities and amino acids increasing in the meat. Zhang et al. (2016) investigated the antimicrobial effects of several different spices on the chicken for 15 days and reported same results.
| Table 2. The results of the measurement pH values | ||||
| Treatment | Storage time (day) | |||
| 0 | 3 | 7 | ||
| 1 | 5.9700±0.042ab | 6.2750±0.035c | 6.8750±0.035f | |
| 2 | 5.9700±0.042ab | 6.0500±0.049b | 6.7150±0.049e | |
| 3 | 5.9700±0.042ab | 5.9700±0.042ab | 6.5100±0.056d | |
| 4 | 5.9700±0.042ab | 5.8600±0.056a* | 6.2550±0.063c | |
| *Treatments with common letters are not significantly different at the one percent level. | ||||
Peroxide value:
The results from the analysis on the peroxide values of lipids from treatments during refrigeration are shown in Table 3. Primary oxidation of lipids during refrigeration storage of treatments measured by peroxide values (Li et al., 2012). Peroxides are compounds without taste and odor and are not recognized by consumers in foods (Özyurt et al., 2012). In this study peroxide values in all treatment groups increased gradually during the storage. This trend was similar to the work of Gómez-Estaca et al. (2007).
The analysis of peroxide data indicated that the peroxide values were significantly affected by treatment contain 3% clove extract throughout refrigeration (P< 0.01). All sample started with low peroxide values of 1.2550±0.077 meq/kg. After 72h, the sample with 3% clove extract presented lower peroxide value than all of the treatments. These results are agreement with the study of Özyurt et al. (2012). So an excellent antioxidant source can prevent lipid oxidation by scavenging free radicals to terminate the free radical chains is clove extract (Özyurt et al., 2012). The highest peroxide value was showed in control treatment after 7 days of refrigeration (P > 0.01). Preventing of lipid oxidation are increased with increasing the amounts of clove extract (Chaijan et al., 2005; Gómez-Estaca et al., 2007). At the end of storage time, the best antioxidant activity belongs to 3% clove extract with peroxide value of 1.5600±0.084 meq/kg that was in agreement with other studies (Gómez-Estaca et al., 2007; Vidya and Srikar, 1996).
| Table 3. The results of the measurement peroxide values (meq/kg) | ||||
| Treatment | Storage time (day) | |||
| 0 | 3 | 7 | ||
| 1 | 1.2550±0.077b | 2.7550±0.077e | 3.4300±0.056f | |
| 2 | 1.2550±0.077b | 1.5750±0.106c | 2.7600±0.084e | |
| 3 | 1.2550±0.077b | 1.5550±0.077c | 2.4600±0.084d | |
| 4 | 1.2550±0.077b | 0.9450±0.056a* | 1.5600±0.084c | |
| *Treatments with common letters are not significantly different at the one percent level. | ||||
TBA:
The results from the analysis of the oxidative degradation of lipids from treatments during refrigeration, measured by TBA numbers, are shown in Table 4. Thiobarbituric acid value has proved to be valuable indicator to assess the degree of lipid oxidation during the food storage time (Barbosa-Pereira et al.; 2013; Gao et al., 2014; Ojagh et al., 2010). In this study, TBA values in all treatment groups increased gradually during the storage. This trend was similar to the work of Kumar and Tanwar (2011). The analysis of TBA data indicated that the TBA values were significantly affected by treatment contain 3% clove extract throughout refrigeration (P< 0.01). All sample started with low TBA values of 0.4340 mg MDA/kg after 72h, the sample with 3% CE presented lower TBA value than all of the treatments. These results are agreement with the study of Iheagwara (2013), and Kumar and Tanwar (2011). Clove extract can be an excellent antioxidant, prevent lipid oxidation by scavenging free radicals to terminate the free radical chains and has been widely used in the preservation of food items such as vegetable oils, animal fats (Aubourg, 1993; Iheagwara, 2013; Kim et al., 2013). The highest TBA value was showed in control treatment after 7 days of refrigeration (P > 0.01) (Kumar and Tanwar, 2011). Preventing of lipid oxidation in combined treatment was increased with increasing the amount of clove extract. At the end of storage time, the lowest TBA belong to the treatments containing 3% clove extract with TBA of 0.3985 mg MDA/kg chicken fillet meat.
| Table 4. The results of the measurement TBA values (mg MDA/kg) | ||||
| Treatment | Storage time (day) | |||
| 0 | 3 | 7 | ||
| 1 | 0.4340±0.018bc | 0.8080±0.022f | 1.2710±0.026g | |
| 2 | 0.4340±0.018bc | 1.4600±0.028c | 0.6615±0.019e | |
| 3 | 0.4340±0.018bc | 0.3710±0.026b | 0.5295±0.019d | |
| 4 | 0.4340±0.018bc | 0.2080±0.016a* | 0.3985±0.019bc | |
| *Treatments with common letters are not significantly different at the one percent level. | ||||
TVN:
The results of TVN from treatments during refrigeration are shown in Table 5. TVN index includes trimethylamine and dimethylamine, ammoniac and other volatile compounds amines in meat are associated with spoilage of meat products and it’s increasing relate to the activity of spoilage bacteria (Ojagh et al., 2010). In this experiment on zero-day, the initial amount of TVN equal to 14 mg per 100 g sample. According to the veterinary regulation system in Iran, the permissible content of volatile nitrogen bases in chicken 28 milligrams per 100 grams. By the end of the maintenance period, the amounts of volatile nitrogen treatments increased significantly, but treatments containing extracts showed fewer total volatile nitrogen. This trend was similar to the work of Fan et al. (2008) that had much lower TVN in treatments containing oregano extract. The highest TVN value was observed in the control treatment after 7 days of refrigeration that it had a significant difference with other treatments (P > 0.01).
| Table 5. The results of the measurement TVN (mg/100g) | ||||
| Treatment | Storage time (day) | |||
| 0 | 3 | 7 | ||
| 1 | 14±1.131a* | 23.0500 ±1.060de | 33.5000±1.838g | |
| 2 | 14±1.131a | 20.6000±0.565cd | 28.6500±0.777f | |
| 3 | 14±1.131a | 19.2500±0.494c | 24.2500±0.777e | |
| 4 | 14±1.131a | 15.1000±0.424ab | 17.6500±0.777bc | |
| *Treatments with common letters are not significantly different at the one percent level. | ||||
3.4. Antimicrobial activity
The results of microbiological analyses are illustrated in Table 6 and Table 7. The initial contamination of the chicken fillet in terms of aerobic plate count (APC) of bacteria and psychrotrophic bacteria were 4.72 and 4.38 log CFU/gr, respectively. The initial population of APC and Psychrotrophic bacteria in the control group increased during the storage period and reached 9.42 and 9.10 log CFU/gr.
The highest amounts of total count (APC) of bacteria and Psychrotrophic bacteria were demonstrated in control treatment after 7 days that had a significant difference with other treatments (P< 0.01).
Similar results were obtained by Govaris et al. (2010) and treatment with 3% clove extract was more effective than others (P< 0.01).
Results clearly demonstrated that clove extract has strong inhibitory effect on Psychrotrophic bacteria and total count growth. It was in accordance with previous reports of Li et al. (2012), who reported that microbial deterioration was delayed about 5 days in crucian carp containing natural extract. Moreover, treatment with 3% clove extract had the lowest counts throughout the storage and showed good results as compared to the control. Similar results were obtained by (Abdollahzadeh et al. (2014); Razavi-Rohani et al. (2011)), and Govaris et al. (2010), who showed that treatment contains antimicrobial agent have good results.
| Table 6. The results of the measurement aerobic plate count (log CFU/gr) | ||||
| Treatment | Storage time (day) | |||
| 0 | 3 | 7 | ||
| 1 | 4.72±0.01b | 7.25±0.03e | 9.41±0.02h | |
| 2 | 4.72±0.01b | 6.69±0.01d | 8.33±0.02g | |
| 3 | 4.72± 0.01b | 6.47±0.08c | 7.95±0.01f | |
| 4 | 4.72±0.01b | 4.11±0.04a* | 7.29±0.02e | |
| *Treatments with common letters are not significantly different at the one percent level. | ||||
| Table 7. The results of the measurement psychrotrophic bacteria (log CFU/gr) | ||||
| Treatment | Storage time (day) | |||
| 0 | 3 | 7 | ||
| 1 | 4.38±0.03b | 7.04±1.06e | 9.10±1.83h | |
| 2 | 4.38±0.03b | 6.70±0.07d | 8.46±0.02g | |
| 3 | 4.38±0.03b | 6.41±0.10c | 7.90±0.04f | |
| 4 | 4.38±0.03b | 4.00±0.00a* | 7.07±0.03e | |
| *Treatments with common letters are not significantly different at the one percent level. | ||||
3.6. Sensory evaluation
The results showed that there is no significant difference between the color of control treatment and other treatments. Also, it was not seen any significant difference between taste and odor scores of control treatment and other treatments of chicken fillet. According to the overall acceptability, results showed no significant differences between the control treatment and other treatments of chicken fillet. There is a significantly different (0.05>P) between the tissue of control treatment and other treatments. The tissue of chicken fillet treated with 3% clove extract was the best and control treatment was the worst. The highest sensory evaluated scores assessed in this research for 3% clove extract treatment.
4. Conclusions
This research showed that clove extract could effectively inhibit lipid oxidation and microbial growth, and could improve sensory attributes, and could extend the shelf life of chicken fillet. Among the treatments tested, the treatment contains 3% clove extract proved the most effective treatment to inhibit lipid oxidation and microbial growth in the chicken fillet. As a consequence, ethanolic clove extract might be considered as effective tools in preventing the quality degradation of chicken fillet, resulting in an extension of their shelf life and our findings suggest that the use of coating methods in combination with natural extract and evaluate the function of these coatings is the effective way to increase the lipid oxidation and the antimicrobial activity in chicken fillet and others meat products.
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